Nervonic acid (cis-tetracos-15-enoic acid; 24:1) is a very long chain fatty acid (VLCFA). There is an increasing interest in production of nervonic acid (24:1 Δ15) for pharmaceutical and industrial applications (1, 2, 3). Nervonate plays a part in the biosynthesis of nerve cell myelin and it is found in sphingolipids of white matter in the human brain (and that of mammals). In diseases involving demyelination such as adrenoleucodistrophy (ALD) and multiple sclerosis (MS), there is a marked reduction of nervonic acid levels in sphingolipids. The administration of nervonic acid to sufferers of these diseases to alleviate the symptoms has been described (4). Nervonic acid has been investigated as a raw material in the pharmaceutical industry for production of medication used for symptomatic treatment of MS (5).
Despite the fundamental lack of understanding of the complexity and inter-relationships of many factors in human brain cells, there is an advantage in providing a supplement of nervonic acid in the diets of children. It is used in the food industry as a supplement to baby and infant formulas/food products (1). It seems to be beneficial to administer nervonic acid to adults whose nervonic acid levels are generally taken to be “normal”, in particular women who intend to be pregnant, are pregnant or lactating (1).
Recently, there has been a strong interest from the University of Guelph and their associates in the Dairy Marketing Board and also at Martek, in high nervonate seed oils. More specifically, the University of Guelph has disclosed a use of nervonate-containing oil in cattle feed to improve the nutritional/health benefit qualities of expressed milk for human consumption (WO 2005036981, published Apr. 28, 2005). Again, the context is in enhanced neural development and as a prophylactic against neurodegenerative diseases. Clearly, an engineered seed oil high in nervonic acid could “spark” these lucrative spin-off utilities. Bioriginal Food and Science Corp. of Saskatoon has also expressed interest in high nervonate seed oil projects.
There are only a few species, most in the Brassicaceae, with high amounts of nervonic acid in their seed oil (6) but only Lunaria annua (syn. Lunaria biennis L; honesty or money plant) is grown as a niche crop. Honesty seed oil has 36-48% erucic but only 14 to 25% nervonic acid (5; our analyses). The oil itself without any chemical modification has been used on a small scale as an industrial lubricant (7, 8). However, this plant is a biennial. Seed yields between 1000 and 2000 kg/ha and an oil content of approximately 30% are low for a crop that needs a growing period of two years before harvest. Seed shattering is also a problem. Thus, it is uneconomical to grow L. annua as a major source of erucic acid or nervonic acid, even with set-aside payments (9). Breeding programs are ongoing in Europe financed by companies CPRO-DLO, VNK and CEBECO from the Netherlands, ADAS from UK, SIA from Spain and DKFZ from Germany to develop L. annua annual types. Although this research effort is progressing well, considerable effort will still be required to develop this crop for commercial use (10).
A Brassica species of special interest which we have identified for the first time to use for high nervonate technology is Cardamine graeca L or bittercress. Cardamine seed oil has from 9 to 10% erucic acid and from 43 to 54% nervonic acid (results from our analyses; 11). It is a small Mediterranean plant found growing on forest floors with red soil (terra rossa) in well sheltered areas. Because of the high level of nervonic acid in the seed oil, we have focused on this plant species and the seed-specific elongase gene FAE involved in biosynthesis of very long chain monounsaturated fatty acids (VLCMFAs). By expressing FAE in Brassicaceae we hope to develop edible oils enriched in nervonic acid which could be of interest to the food industry, for human consumption, or in the nutraceutical industry and as an additive in dairy-livestock feed to produce milk enriched in nervonic acid (12). Such oils should be high in nervonic acid but low (<10%) in erucic acid for acceptability in these markets.
VLCMFAs are synthesized outside the plastid by a membrane bound fatty acid elongation complex (elongase) using acyl-CoA substrates. The first reaction of elongation involves condensation of malonyl-CoA with a long chain substrate producing a 3-ketoacyl-CoA. Subsequent reactions are reduction to a 3-hydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a second reduction to form the elongated acyl-CoA. The 3-ketoacyl-CoA synthase (KCS) catalyzing the condensation reaction plays a key role in determining the chain length of fatty acid products found in seed oils and is the rate-limiting enzyme for seed VLCFMA production. The composition of the fatty acyl-CoA pool available for elongation and the presence and size of the neutral lipid sink are additional important factors influencing the types and levels of VLCFMAs made in particular cells.
Knowledge of the mechanism of elongation and properties of fatty acid elongase condensing enzymes is, in part, limited by their membrane-bound nature. As such they are more difficult to isolate and characterize than soluble condensing enzymes.
To date, increases in the content of some strategic fatty acids have been achieved by introduction of various fatty acid biosynthesis genes in oilseeds. Some examples include:                expression of a medium chain fatty acid thioesterase from California Bay, in Brassicaceae to produce lauric acid (Calgene);        expression of an anti-sense construct to the Δ9 desaturase in Brassicaceae to increase the stearic acid content (Calgene);        use of co-suppression constructs encoding plant microsomal desaturases to increase proportions of oleic acid (DuPont/Cargill); and,        expression of the Arabidopsis FAE1 gene in HEAR to increase the proportion of erucic acid by 10% or more (14).        
There is some information on the FAE gene from L. annua (money plant). Lassner from Calgene stated that the heterologous expression of L. annua FAE in high erucic acid rapeseed (HEAR) apparently resulted in accumulation of approximately 20% nervonic acid in the seed oil (13). However, no data on L. annua FAE nucleotide and/or protein sequences were published and no data on L. annua FAE nucleotide and/or protein sequences were published and no data on experimental procedures were provided. Neither was there any accompanying report of the erucic acid content.
To date, no elongase genes have been isolated from C. graeca and characterized as encoding an elongase to produce nervonic acid. Similarly, not until the present invention has Teesdalia nudicaulis been identified as a source for a gene encoding an FAE for producing oils enriched in eicosenoic acid. To date, there is no published data, to our knowledge, on T. nudicaulis FAE sequence and its utilization.
Commonly owned PCT international patent application PCT/CA2004/002021 filed Nov. 24, 2004 discloses FAE genes cloned from nasturtium and Crambe. An elongase gene (FAE1) from Arabidopsis was cloned and published as James, D. W. Jr., Lim, E., Keller, J., Plooy, I., Ralston, E. and Dooner, H. K., “Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator”. The Plant Cell 7: 309-319 (1995). Other related prior art includes: sequence ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 35, 37, 39, 41 from Jaworski, J. G. and Blacklock, B. J., world patent publication WO0194565 published Dec. 13, 2001; sequence ID NOs: 2, 4, 6, 12, 14, and sequences ID NO: 1, 3, 5, 7, 9, 11 and 13 from Jaworski et al., U.S. Pat. No. 6,307,128 issued Oct. 3, 2001; and, sequence ID NOs: 19, 20, 21, 22, 23 from Kunst and Clemens, world patent publication WO0111061 published Feb. 15, 2001.